Methods to identify swine genetically resistant to F18 E. coli associated diseases

ABSTRACT

The present invention provides non-invasive methods and compositions to differentiate, with a high level of sensitivity and specificity, swine that are genetically susceptible to diseases associated with F18  E. coli  infection, from resistant swine. DNA polymorphisms in the swine alpha (1,2) fucosyltransferase 1 (FUT1) gene were used to differentiate resistant from susceptible swine. The invention includes a polypeptide with amino acid substitutions, encoded by the nucleotide polymorphisms, a molecular diagnostic assay, and a kit for the differentiation, of  E. coli  F18-adhesion resistant, heterozygous (carrier) and homozygous susceptible pigs. The molecular test identifies susceptibility to oedema disease and postweaning diarrhea with high sensitivity and specificity, therefore, is useful to swine breeder in their effort to enhance for resistance. Information on the polymorphisms of the present invention provides insight into causation and treatment of  E. coli  associated intestinal disorders.

This application claims priority to U.S. Provisional Application60/047,181 filed May 20, 1997, now abandoned; PCT/US98/10318, filed May20, 1998; and is a divisional of U.S. Ser. No.09/443,766 filed Nov. 19,1999 now U.S. Pat. No. 6,596,923.

Compositions and non-invasive methods are provided for theidentification of swine genetically resistant to E. coli bacteriasupplied with fimbriae F18. DNA polymorphisms in the swine alpha (1,2)fucosyltransferase (FUT1) gene were identified that differentiateresistant from susceptible swine and provide a diagnostic test usefulfor swine breeders.

A major problem in breeding swine is to keep them disease-free.Intestinal disorders postweaning are a particular problem. A limitednumber of serotypes of toxigenic Escherichia (E. coli) strains are thecausative agents of oedema disease and postweaning diarrhea in swinewhich induce serious economic losses, especially among piglets aged 2 to4 weeks, in swine breeding farms all over the world. The typicalsymptoms of oedema disease are neurological signs such as ataxia,convulsions and paralysis. At post mortem examination, oedema istypically present at characteristic sites such as eyelids and forehead,stomach wall and mesocolon. The diseases are caused by Shiga-liketoxin-II variant and enterotoxins LT, Sta, Stb respectively, produced byE. coli that colonize the surface of the small intestine withouteffecting major morphological changes of the enterocytes (cells in theintestine). Certain types of bacterial E. coli strains, F18, F4 and K88are major lethal villains in this regard. “Oedema disease of pigs is anenterotoxaemia characterized by generalized vascular damage. The latteris caused by a toxin, Shiga-like toxin II variant, produced by certainstrains of E. coli” (Bertschinger et al., 1993). The E. coli aredistinguished by their pili types, a group of adhesive fimbriae that arerelated are designated e.g., K88 or F18 (Vögeli et al., 1997).

Not all swine succumb to E. coli infections. Colonization depends onadherence of the bacteria to the enterocytes which is meditated by thebacterial fimbriae designated e.g., K88 or F18. Susceptibility toadhesion, i.e. expression of receptors in swine for binding thefimbriae, has been shown to be genetically controlled by the host and isinherited as a dominant trait with, in the case of F18, B being thesusceptibility allele and b the resistance allele (Vögeli et al., 1996;Meijerink et al., 1996). The genetic locus for this E. coli F18-receptor(ECF18R) has been mapped to porcine chromosome 6 (SSC6), based on itsclose genetic linkage to the S locus and other loci of the halothane(HAL) linkage group on chromosome 6. The receptor for K88 E. coli is onchromosome 13.

The mechanism for resistance appears to be that intestinal borders inresistant animals are not colonized by E. coli, i.e., the bacteria do noadhere to intestinal walls of resistant swine. Glycoprotein receptors inthe brush border membrane of the intestine were shown to be responsiblefor the differences between adhesive and non-adhesive phenotypes relatedto some E. coli, therefore, the genotype of the host swine determinesresistance. The fimbriated bacteria also have been studied (WO 9413811).

Current methods of identifying swine that are resistant to F18 E. coliassociated diseases are either to 1) collect intestinal samples fromswine at slaughter and perform the microscopic adhesion test, 2)challenge the animals with virulent E. coli (“colonization test”), or 3)perform blood typing of the A-O(S) blood group system. The first twomethods are not practical for identifying resistant animals for use asbreeding stock. Although the blood typing method does identify resistantanimals, the test is unable to determine whether susceptible animals arehomozygous or heterozygous for susceptibility. Knowledge of the genotypeof animals with regard to these alleles (conditions of a gene) isessential to develop a successful breeding program. The purpose of thebreeding program is to produce swine that are resistant to F18 E. coliassociated diseases that decimate stock post-weaning.

In one publication the authors stated, in reference to oedema disease inswine, that “Searches are underway for appropriate genetic markers . . .” (Bertschinger et al., 1993, page 87) and, citing Walters and Sellwood,1982:

-   -   Breeding resistant swine is an attractive method for prevention        of diseases for which an effective prophylaxis is not available.        The feasibility of this approach will depend on the prevalence        of the gene(s) encoding resistance in the pig population,        improved methods for the detection of resistant pigs, and        absence of negative genetic traits co-selected with this        resistance.

A genetic “marker” locus is a coding or non-coding locus that is closeto a genetic locus of interest, but is not necessarily the locus itself.Detectable phenotypes include continuous or discontinuous traits, e.g.restriction length fragment polymorphisms, production traits, bacterialadhesion traits, calorimetric or enzymatic reactions, and antibioticresistance. The S locus controls expression of the A and O blood groupantigens. Swine homozygous recessive at the S locus do not expresseither A or O blood group antigens. A similar condition exists in humansand is due to mutations in the alpha (1,2) facosyltransferase gene whichencodes the human blood group H (Kelly et al., 1994; see also WO9628967). The porcine alpha (1,2) fucosyltransferase gene of swine hasrecently been sequenced (Colney et al., 1996). This gene is very likelythe gene present at the S locus in swine.

The blood group H and Se loci have been mapped genetically andphysically to human chromosome 19q13.3. This region is evolutionarilyconserved, containing genes homologous to the HAL linkage group of genesin pigs. The blood group H encoding gene is the so called FUT1 whereasthe Se gene is equivalent to the FUT2 gene. FUT1 determines H antigenexpression in the erythroid cell lineage, whereas FUT2 regulatesexpression of the H antigen in the secretory epithelia and saliva.Conservation of the FUT1 gene has seen shown in lower mammals such asrat and rabbit, and mRNA expression has been shown in rabbit braintissue and rat colon. In all these species two types of alpha (1,2)fucosyltransferase genes have been reported which are structurally verysimilar to the human FUT1 and FUT2 genes, but in particular the FUT1homologous genes show a species specific expression pattern. In humansthe FUT1 gene is responsible for synthesis of H antigens in theprecursors of erythrocytes. However, in pigs erythrocytes passivelyabsorb H-like antigens from the serum, as is the case for the humanLewis antigens. In pigs all H-like antigens are related to exocrinesecretory tissues, and expression of the FUT2 (Secretor) gene is seen insecretory tissue of other animal species. Therefore, expression of theporcine A-O blood group determinants which cross-react with anti-humanblood group H and A antibodies might be influenced by the FUT2 gene.

Further information about blood groups and E. coli swine diseasesinclude that carbohydrate structures of blood group antigens mediate theadhesion of some pathogenic microorganisms to host tissues, e.g.Helicobacter pylori adhere to Lewis^(b) blood group antigens, and E.coli causing urinary tract infections adhere to blood group P substance.Genes encoding glycosyltransferases that are responsible for theformation of the blood group specific carbohydrate structures,therefore, represent candidate genes for the control of bacterialcolonization by the host. The localization of these genes is in the samechromosomal region as the locus responsible for adhesion/non-adhesion ofF18 positive E. coli in the swine small intestine. Swine do not expressblood group antigens A and O until after weaning, this is the same timethat they become susceptible to disease caused by F18 E. coli.

New methods of diagnosis and treatment are needed for E. coli relatedintestinal diseases in swine. Detection of a genetic mutation wasproposed as a diagnostic test for some swine disorders (malignanthypothermia) (Fujii et al., 1991; U.S. Pat. No. 5,358,649), butpolymorphic markers were not reported for diagnosis. Vaccines to developresistance to E. coli colonization were described (U.S. Pat. No.5,552,144; WO 8604604), but are unlikely to be a preferred method toprevent the E. coli disease because of difficulties in administeringlive vaccine orally to newborn swine, and because of regulatoryrestrictions. Antibiotics are available for treatment, but there is nosuccessful prophylaxis.

SUMMARY OF THE INVENTION

The compositions and non-invasive methods of the present inventionprovide detection and elimination of swine that are susceptible to E.coli associated diseases. A non-invasive method for identifying a swinethat is resistant to intestinal colonization by E. coli F18 includes thefollowing steps: determining whether a genetic polymorphism associatedwith resistance to colonization is in a biological sample from theswine; and inferring that the swine is resistant if the swine ishomozygous for the polymorphism.¹

¹A polymorphism is a change in a nucleotide sequence that exists in apopulation due to mutation.

More particularly, the method is determining in a biological sample fromthe swine whether the nitrogen base at position 307 in the alpha (1,2)fucosyltransferase gene of the swine is only adenine or only guanine;and identifying the swine as resistant if the only nitrogen base atposition 307 is adenine.

To determine whether a polymorphism is present in a biological sample,restriction fragment length polymorphisms are analyzed on a gel thatseparates them by molecular weight. Restriction endonucleases areenzymes that reproducibly cut nucleic acid molecules at specific sites,resulting in nucleic acid fragments of different molecular weights,depending on the location of the cuts.

The invention also relates to a method for breeding swine to beresistant to E. coli associated diseases by selecting for breeding swinethat have a genetic polymorphism in the alpha (1,2) fucosyltransferase 1gene that identifies them as swine that are resistant to E. coli relatedintestinal diseases; and breeding the selected swine.

An aspect of the invention is a DNA molecule which is polymorphic forthe alpha (1,2) fucosyltransferase 1 gene in swine, in particular asequence in accordance with FIG. 1. Other aspects of the invention aremolecules with nucleotide sequences complementary to that of FIG. 1.

An aspect of the invention is an isolated DNA molecule with asubstitution of adenine for guanine in position 307. This molecule mayalso have a substitution of adenine for guanine in position 857. Otherisolated DNA molecules of the present invention include those with amutation at nucleotide position 229 of the sequence of FIG. 1, whereinthe codon CTT is changed to TTT, encoding for the amino acidphenylalanine instead of leucine. A mutation at nucleotide position 714is from GAT→GAC, but there is no accompanying amino acid substitution inthe encoded product.

Polypeptides encoded by the DNA molecules of the present invention andhaving alpha (1,2) fucosyltransferase activity are also aspects of theinvention.

A molecular assay for detecting E. coli F18 receptors in swine is to (a)isolate DNA from porcine nucleated cells; (b) amplify the DNA in apolymerase chain reaction (PCR) using oligonucleotides as primers whichare complementary to a DNA sequence of the porcine alpha (1,2)fucosyltransferase gene 1; (c) perform a restriction enzyme digest withat least one restriction enzyme e.g., CfoI; (d) separate the resultingfragments by gel electrophoresis; and (e) determine the respectivenumbers and lengths of fragments on the gel; and (f) determine from thenumbers and length of fragments, which receptors are present in theporcine cells. Use of the larger amplified fragments disclosed hereinfor restriction length polymorphism analysis (RFLP), rather than smallerfragments, is less expensive because the DNA bands can be run on agarosegels of relatively low concentration. Also, to produce some of thefragments, only one restriction enzyme is needed for a constantrestriction site adjacent to the variable diagnostic site.

A kit for detecting polymorphisms associated with E. coli F18 receptorsuses oligonucleotides in separate containers which are complementary toa DNA sequence of the porcine alpha (1,2) fucosyltransferase gene 1 thatdistinguishes resistant from sensitive swine. The test can be performedon swine of any age.

The polymorphisms are also useful to develop drugs to treat swine thathave E. coli-associated disease. A mutated form of porcine alpha 1,2fucosyltransferase could interfere with the normal enzyme, preventing itfrom producing the intestinal receptor for F18.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence (FUT1) (below) and the predictedamino acid sequence (above) of the swine α1→2fucosyltransferasepolymorphism of the present invention using the one-letter amino acidcode. The solid double line below the amino acid sequences (=) is theputative transmembrane region; the dotted line below the amino acidsequence shows three potential N-linked glycosylation sites ( . . . ).

□ is where an adenine (A) is substituted for guanine (G) in resistantswine.

* Indicates the termination codon.

Abbreviations for the amino acid residues are as follows: A, Ala; C,Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M,Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; andY, Tyr.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Molecular analysis of DNA polymorphisms associated with resistance ofswine to E. coli associated diseases facilitated diagnostic assays toselect resistant pigs for breeding. Resistant pigs differ from sensitivepigs at the E. coli F18 receptor locus as identified by the polymorphicmarkers of the present invention.

The present invention provides non-invasive methods and compositions todistinguish with a high level of sensitivity and specificity, swine thatare genetically susceptible to diseases associated with F18 E. coliinfection from resistant swine. A DNA polymorphism in the swine alpha(1,2) fucosyltransferase (FUT1) gene was identified that differentiatesresistant from susceptible swine. The polymorphism arose by a mutation(change) in a nucleotide sequence leading to a new allele. An allele isa condition of a gene. In a population there may be many alleles of agene differing by nitrogen base substitutions, presumably caused bymutations in an ancestral DNA molecule. The coexistance in a populationof more than one allele (sometimes referred to as a “variant”) is calleda genetic polymorphism. Loci at which more than one allele may exist asapparently stable components of a population, is a polymorphic locus.Usually, one of the polymorphic loci is at a low frequency in thepopulation.

As determined from a biological sample, preferably blood, the resistantswine have a polymorphism in their genomes in which the only basedetected at position 307 (see FIG. 1) in the nucleotide sequence isadenine, whereas the base in the same position in homozygous susceptibleswine is guanine. Heterozygous swine will show both types of DNA andwill be susceptible. The polymorphism is a variation of a porcine genesequence (Cohney et al., 1996).

Genetic linkage analysis was performed on families of swine and geneticassociations between polymorphisms in FUT1 and disease resistance inoutbreed swine were determined. According to the present invention,polymorphisms have been found in the alpha (1,2) fucosyltransferase 1gene (FUT1). A polymorphism that has a single nucleotide basesubstitution at position 307 was used to establish a close linkagebetween the fucosyltransferase gene and the S-system, the ECF18R locusand other loci of the HAL linkage group.

The detection of the close linkage of the mutation at FUT1 and ECF18Rallowed a molecular test to be developed for the identification of E.coli F18 adhesion resistant, heterozygous (carrier) and homozygoussusceptible pigs. This diagnostic test identifies, with high sensitivityand specificity, pigs that are susceptible to oedema disease andpostweaning diarrhea. The incidence of the polymorphisms of the presentinvention differs among swine breeds. Vögeli, et al. (1997) presentedfrequencies of the M307 allele in 5 pig breeds from herds that were notrelated to one another. The availability of the diagnostic test for thepolymorphism of the present invention provides breeders with theopportunity to effectively eliminate the ECF18R susceptible allele fromtheir swine herd, thereby eliminating a prerequisite for E. coli F18bacterial adhesion causing oedema disease and postweaning diarrhea.

The present invention further includes nucleotide sequences that arevariants of a sequence of the alpha (1,2) fucosyltransferase gene 1representing the various polymorphisms at bp 307, (SEQ ID NO: 1) anddiagnostic molecular based kits to identify polymorphisms in the alpha(1,2) fucosyltransferase gene.

In order to obtain candidate genes for the E. coli F18 receptor locus(ECF18R) 5 cosmids and one genomic clone containing the gene wereisolated containing the alpha (1,2) fucosyltransferase genes, FUT1 andFUT2 (Meijerink et al., 1997), from a porcine genomic library. Mappingby fluorescence in situ hybridization placed all these clones in bandq11 of porcine chromosome 6 (SSC6q11). Sequence analysis of the cosmidsresulted in the characterization of (a) an open reading frame (ORF),1098 base pairs in length, that is 82.3% identical to the human FUT1sequence, and (b) a second ORF, 1023 base pairs in length which is 85%identical to human FUT2 sequence. The FUT1 and FUT2 loci therefore seemto be porcine equivalents of the human blood group H and the Secretorlocus. Direct sequencing of the two ORF's in swine either susceptible orresistant to adhesion and colonization by F18 fimbriated E. coli(ECF18R) revealed two polymorphisms at base pair 307 (M307) and basepair 857 (M857) of the FUT1 ORF. The nucleotide positions are numberedfrom ATG (encoding methionine). Analysis of these mutations in 34matings of Landrace families with 221 progeny showed close linkage withthe locus controlling resistance and susceptibility to E. coli F18adhesion and colonization in the small intestine (ECF18R) and with thelocus of the blood group inhibitor S. Therefore, the M307 mutation is agood marker for marker-assisted selection of E. coli F18 adhesionresistant animals. Another mutation at nucleotide position 229 was foundleading to a polymorphism in which the codon encoding leucine (CTT) waschanged to TTT (encoding phenylalanine). A mutation at position 714(GAT→GAC) (encoding aspartic acid) did not produce an amino acidsubstitution. No polymorphisms were identified in FUT2 thatdifferentiated susceptible and resistant pigs.

EXAMPLES

The following examples provide embodiments of the invention.

Example 1 An Assay For Resistant Swine

The polymorphisms of the present invention are easily identified usingPCR-RFLP tests. One embodiment of the tests used a 160 bp fragment ofporcine alpha (1,2) fucosyltransferase 1 amplified using PCR with thefollowing primers; 5′CCAACGCCTCCGATTCCTGT3′ and5′GTGCATGGCAGGCTGGATGA3′. (SEQ ID NO :2) Preferred PCR conditions forthis embodiment are 25 cycles at the following times and temperatures:94° C., 30 sec; 60° C., 45 sec; 72° C., 90 sec. The amplified DNA fromresistant swine was digested by the restriction enzyme Hgal, but was notdigested by the restriction enzyme HinPI. The amplified DNA fromhomozygous susceptible swine was digested by the restriction enzymeHinPI. The amplified DNA from heterozygous susceptible swine waspartially digested by both enzymes.

Alternatively, DNA was isolated from porcine nucleated cells accordingto standard procedures. Direct sequencing of porcine FUT1 and FUT2sequences and their flanking regions in animals of different ECF18Rgenotype (Bb, bb) resulted in the identification of two G→A transitionsat positions 307 and 857 (termed M307 and M857, respectively) of theFUT1 ORF. The M307 transition eliminates a restriction site for CfoI.Amplification of DNA isolated from porcine nucleated cells was preformedaccording to standard procedures with primers P6 and P11 (3 min at 95°C., 30 cycles of 30 sec at 95° C., 30 sec at 56° C. and 30 sec at 72°C., followed by a 7 min final extension 72° C.) followed by CfoIdigestion and separation on a 3% agarose gel resulted in a restrictionfragment length polymorphism (RFLP). Homozygous M307^(AA) animals showed2 bands. Homozygous M307^(GG) animals showed 93-, 241- and 87 bpfragments. Heterozygous animals showed all four fragments.

Example 2 Sensitivity and Specificity Of An Assay Using Alpha (1,2)Fucosyltransferase In Detecting Swine Resistant to F18 E. coli

A study was conducted to determine the association between diseaseresistance and the polymorphism at position 307 of the FUT1 gene. 183weaned swine (ranging in ages 2-6 months) were obtained from sixdifferent breeding herds. Only one of these herds was known to containresistant animals before the start of the study, and this herd is knownto have a high incidence of porcine stress syndrome. The other 5 herdshad no evidence of porcine stress syndrome, and the incidence of diseaseresistance was unknown. Swine from each herd were randomly selected,humanely euthanized and spleens and samples of small intestine wereremoved. DNA was extracted from splenic tissue and used in a PCR-RFLPassay described in Example 1. Intestinal cells were purified by scrapingthe mucosal surface off the intestine, lysing the cells in a hypotonicEPTA solution and washing by centrifugation. The purified intestinalcell brush borders were incubated with F18 E. coli. This mixture wasexamined by phase contrast microscopy. This assay determined if swinewere susceptible (intestinal samples had adhering bacteria) or resistant(intestinal samples had no adhering bacteria). The PCR-RFLP assay forthe polymorphism correlated with the bacteria-intestinal cell bindingassay in 53 of 53 resistant swine and 128 of 130 susceptible swine. Twoswine that were determined susceptible using the bacteria-intestinalcell binding assay were incorrectly predicted to be resistant using thePCR-RFLP assay. Two of the six herds examined contained resistant pigs,while only one herd had porcine stress syndrome, demonstrating that thePCR-RFLP assay can identify disease resistant animals in animals that donot have porcine stress syndrome.

Example 3 Localization of FUT1 on Chromosome 6 (SSC6)

Cosmids ETHs1, −s2, −s3, −s4 and −s6 were identified after screening ofthe cosmid library with a FUT1 nucleotide probe obtained from porcinegenomic DNA with primers P7 and P10 and were mapped by FISH and DISC-PCRto chromosome 6 in band q11.

Example 4 Identification of the Porcine FUT1 ORF

Hybridizing KspI, EcoRI and KspI/EcoRI cosmid digests with radiolabelledporcine FUT1 fragments P6-P11 and P7-P10 for Southern blot analysisrevealed identical autoradiography signals for ETHs2, −s4 and −s6,whereas different signals were obtained from cosmids ETHs1 and −s3. Fromcosmid ETHs2 KspI, subclones 940 bp and 6.2 kb in length were isolated,corresponding to the estimated length of hybridizing KspI fragments onthe Southern blot. The sequence results of both subclones were combinedto yield a 1501 bp sequence, which was in agreement with results ofdirect sequencing of genomic PCR products. The 1501 bp sequence containsan open reading frame (ORF) of 1098 bp corresponding to the human FUT1ORF, with 82.3% nucleotide and 80.8% amino acid identity. The ORFencodes a polypeptide.

Example 5 Identification of a Porcine FUT2 and a Pseudogene FUT1

ETHs1 has one DNA fragment (2.7 kb) that hybridizes to FUT1 sequences,whereas ETHs3 has two (2.7 kb and 8.2 kb). Subcloning and partialsequencing of the 2.7 kb EcoRI fragment of ETHs1 and −s3 confirmed thatthese two fragments are identical. The sequence is highly similar to thehuman FUT2 but shows several changes in the NH₂— and —COOH terminalregions. These changes lead to frame shifts that are not compatible witha conserved ORF, therefore an assumption is that the sequence obtainedfrom the 2.7 kb fragment represents a pseudogene (FUT2P). Aftersubcloning of ETHs3 BamHI digests, the hybridizing sequences containedin the 8.2 kb EcoRI fragment were identified. The sequence of thesubclones obtained represents a 1023 bp ORF and is 85% identical at thenucleotide- and 83%-identical at the amino acid level to the human FUT2sequence. Many differences in the NH₂— and —COOH terminal regions wereobserved between the porcine FUT2 sequence and the FUT2P sequencederived from the 2.7 kb fragment. The predicted amino acid sequencecorresponds to the partially determined amino acid sequence of theporcine Secretor enzyme (Thurin and Blaszczyk-Thurin, 1995). The porcineFUT1, FUT2, and FUTP sequences obtained were submitted to GenBank andhave accession numbers U70883, U70881 and U70882, respectively. The FUT1and FUT2 genes have highly homologous sequences. This has to beconsidered in, for example, primer development. Furthermore, FUT1 andFUT2 enzyme activity need to be differentiated in further studies.

Example 6 Identification of M307 and M857 Mutations and Characterizationof M307

DNA was isolated from porcine nucleated cells according to standardprocedures. Direct sequencing of porcine FUT1 and FUT2 sequences andtheir flanking regions in animals of different ECF18R genotypes (Bb, bb)resulted in the identification of two G→A transitions at positions 307and 857 (termed M307 and M857, respectively) of the FUT1 ORF. The M307transition eliminates a restriction site for the enzyme CfoI.Amplification of DNA isolated from porcine nucleated cells was performedaccording to standard procedures with primers P6 and P 11 (3 min at 95°C., 30 cycles of 30 sec at 95° C., 30 sec at 56° C. and 30 sec at 72°C., followed by a 7 min final extension at 72° C.) followed by CfoIdigestion and separation on a 3% agarose gel resulted in a restrictionfragment length polymorphism (RFLP). Homozygous M307^(AA) animals showed2 bands (93- and 328-bp fragments). Homozygous M307^(GG) animals showed87-, 93-, an 241-bp fragments. Heterozygous animals showed all fourfragments.

Example 7 Characterization of Mutation M857

The M857 mutation is a transition that eliminates an AciI site. PrimerPBEST was designed to mismatch two additional AciI sites at positions866 and 872. PCR with primers P7 and PBEST (3 min at 95° C., 30 cyclesof 30 sec at 95° C., 30 sec at 56° C. and 30 sec at 72° C., followed bya 7 min final extension at 72° C.) followed by AciI digestion enablesPCR-RFLP analysis on a 3% agarose gel. Homozygous M857^(AA) animals showa 174 bp fragment while amplification products of M₈₅₇ ^(GG) animalsshow 136- and 38-bp fragments.

Example 8 Genetic Mapping of the FUT1 Gene

In Landrace swine families, recombination events between M307 and theloci of the HAL linkage group (S, ECF18R, RYR1, GPI, PGD) revealedrecombination fractions Θ<0.04 (Table 2). The lodscores Z for theoverall recombination fractions were between 24.5 and 50.6, showingstrong evidence for linkage between these loci. These data allow geneticmapping of the FUT1 gene to the HAL linkage group in close proximity ofS and ECF18R which are both influenced by FUT1. In experimental Landracefamilies, allelic association was found between ECF18R and RYR1. Anexcess of genotypes RYR1^(TT) at position 1843 in RYR1 (halothanesusceptible genotype) was observed among pigs resistant to oedemadisease and postweaning diarrhea (genotype ECF18R^(b/b)) (Table 3). Thisallelic association is a result of linkage disequilibrium, that is,deviation of observed haplotype frequencies from expected haplotypefrequencies under independent assortment of alleles. Therefore, linkagedisequilibrium designates a non random association of alleles belongingto linked loci. Owing to low recombination rates however, no locus ordercould be determined as being significantly better than others.

Example 9 Association of M307^(A) With ECF18^(b) And M307^(G) WithECF18R^(B)

In Landrace (SL) and Large White (LW) parental pigs, ECF18R^(b) (theoedema and postweaning diarrhea resistance allele) is 100% associatedwith M307^(A), and ECF18R^(B) (the oedema and postweaning diarrheasusceptibility allele) is 100% associated with M307^(G) (whereinA=adenine; G=guanine). In SL pigs 88% (30/34) of S^(s) accounted for allECF18R^(b) and M307^(A) haplotypes, respectively. The correspondingvalues for both the S^(s)-ECF18R^(b) and S^(s)-M307^(A) haplotypes were82% (9/11) in Large White pigs. In the experimental SL families, theoccurrence of the M857^(A) allele at the FUT1 locus was low, and evenabsent, in LW pigs. Therefore, a significant gametic association was notobserved between the alleles of M857 and the alleles of the flankinggenes. The G→A transitions at positions FUT1 307 and FUT1 857 were foundwith variable frequencies also in Duroc, Hampshire and Pietrain pigs,making it likely that those transitions also occur in other pig breeds.

Example 10 Distribution of FUT1 Genotypes

Table 4 shows that the distribution of FUT1 genotypes at nucleotideposition 307 among ECF18R types was significantly different from theexpected ratio under a hypothesis that the two are independent. Of the119 oedema disease and postweaning diarrhea resistant ECF18R^(b/b)animals, 118 were determined to have the genotype M307^(AA) in theDNA-based test. One resistant animal had the genotype M307^(A/G). Of the131 susceptible pigs, 130 were M307^(A/G) or M307^(G/G). One animal,susceptible to E. coli adhesion, was shown to be homozygous M307^(A/A)by the DNA-test. The data from this example and example 2, together withpast studies suggested that the FUT1 gene is the gene present at the Slocus in swine and the ECF18 locus. While 4 animals in this example andexample 2 contradict this hypothesis, it is probable these animals wereincorrectly phenotyped in regards to disease resistance/susceptibility.

Example 11 Amino Acid Exchanges in Alpha (1,2) Fucosyltransferase

The G→changes at bp +307 and bp +857 of the alpha (1,2)fucosyltransferase gene 1 results in a predicted amino acid substitutionof threonine (neutral-polar) instead of alanine (neutral-nonpolar) andglutamine (neutral-polar) instead of arginine (basic), respectivelywhich may have functional consequences in the encoded product. AC→Tchange at bp 229 results in an amino acid substitution of leucine(neutral-nonpolar) instead of phenylalanine (neutral-nonpolar).

TABLE 1 Sequences Of Forward-(F) And Reverse-(R) Primers And TheirRelative Position to the Porcine FUT1 and FUT2 Start Codons² Primer namePrimer Sequence Position FUT1 P6 (R) 5′-CTTCAGCCAGGGCTCCTTTAAG-3′ +489(SEQ ID NO: 3) FUT1 P7 (F) 5′-TTACCTCCAGCAGGCTATGGAC-3′ +720 (SEQ ID NO:4) FUT1 P10 (R) 5′-TCCAGAGTGGAGACAAGTCTGC-3′ +1082 (SEQ ID NO: 5) FUT1P11 (F) 5′-CTGCCTGAACGTCTATCAAGATC-3′ +69 (SEQ ID NO: 6) FUT1 P16 (F)5′-AGAGTTTCCTCATGCCCACAGG-3′ −90 (SEQ ID NO: 7) FUT1 P18 (R)5′-CTGCTACAGGACCACCAGCATC-3′ +1203 (SEQ ID NO: 8) FUT1 PBEST5′-ACCAGCAGCGCAAAGTCCCTGAC +893 (R) GGGCACGGCCTC-3′ (SEQ ID NO: 9) FUT2P16 (R) 5′-CTCCCTGTGCCTTGGAAGTGAT-3′ +1094 (SEQ ID NO: 10) FUT2 P17 (F)5′-AACTGCACTGCCAGCTTCATGC-3′ −83 (SEQ ID NO: 11) ²Primers FUT1 P10 andFUT1 P11 are derived from the human FUT1 gene.

²Primers FUT1 P10 and FUT1 P11 are derived from the human FUT1 gene.

TABLE 2 Overall Recombination Fractions (θ), Lodscores (Z) And Number OfInformative Animals (N) For M307 And Loci Of The HAL Linkage Group InThe Landrace Experimental Population Locus pair N θ Z S-ECF18R 183 0.0150.6 M307-S 183 0.01 50.6 M307-ECF18R 216 0.01 57.1 M307-RYR1 198 0.0247.2 M307-GP1 147 0.03 34.2 M307-PGD 147 0.04 24.5

TABLE 3 Haplotype Frequencies At the Four Loci (S-FUT-1 (M307, M587)-ECF18R-RYR1) In the Landrace (SL) Experimental Population And RandomlySelected Large White (LW) Pigs. Haplotype³ at S, FUT1 (M307, M857),ECF18R, Frequency⁴ Breed RYR1 (number) SL sAGbT 70 (28) sAGbC  5 (2)sGGBC 15 (6) sGABC 10 (4) LW sAGbC 56 (9) sGGBC 31 (5) sGGBC 13 (2) ³S:Suppressor locus for A and O blood types (S and s). FUT1 (M307):alteration of adenine (A) to guanine (G) at nucleotide 307 of the alpha(1,2) fucosyltransferase (FUT1) gene. FUT1 (M857): alteration of adenine(A) to guanine (G) at nucleotide 857 of the FUT1 gene. ECF18R: E. coliF18 receptor. The dominant susceptible allele is indicated by B and theresistant allele by b. RYR1: skeletal muscle ryanodine receptor. C(cytosine) is the dominant resistant and T (thymine) the # susceptibleallele for malignant hyperthermia. ⁴Haplotype frequencies in % andabsolute number of haplotypes between brackets.

TABLE 4 Distribution Of The Genotypes, Tetrachoric Correlation (R) AndSignificance Of The Association (X² And w × X²) Of The AssociatedPolymorphic FUT1 (M307) And ECF18R Loci In Landrace (SL) ExperimentalPopulation And Randomly Selected Large White (LW) Swine. Breed LocusFUT1/M307 A/A r X² X² × w⁵ Genotype A/G SL ECF18R⁶ b/b 1 113 0.98 213.142.6*** B/B 106 1 A/G Genotype (A/G) G/G LW ECF19R⁶ b/b 0 5 1.00 29.011.6*** B/b, B/B 24 0 ⁵A weight factor of w = 0.2 (SL) and 0.4 (LW) wasapplied to correct for the lack of precision resulting from inclusion ofrelated animals in the data, according to Cotterman (1947). ***p >0.001. ⁶Animals of genotype b/b at the ECF18R locus are resistant andthose of genotype B/b and B/B are susceptible to adhesion of F18ab E.coli bacteria.Methods1. Primers

Primers derived from the human FUT1 gene were used for the amplificationof its porcine counterpart from genomic DNA. From the resulting porcinesequences specific primers were designed which were used in furtheramplification and sequencing reactions (Table 1).

2. Screening of a Porcine Genomic Library

Porcine genomic libraries were screened with either a porcine FUT1 probeobtained with primers P7 and p10 or a porcine FUT1 cDNA. A porcinegenomic library, constructed in SuperCos 1 (Stratagene, LaJolla, Calif.,USA), was screened with an α³²P dATP labeled (Prime It II, Stratagene)FUT1 probe obtained from porcine genomic DNA with primers P7 and P10.After hybridization of replica filters at 42° C. for 15 h (50%formamide, 6×SSC, 5×Denhardt's, 0.5% SDS, 0.1 mg/ml Salmon Sperm) andwashing twice at 65° C. for 30 min. (1×SSC, 0.1% SDS), positive colonieswere identified after exposure (15 h, −80° C.) to X-ray film.

3. In Situ Hybridization of Porcine Metaphase Chromosomes

Cosmid clones ETHs1, ETHs2, ETHs3, ETHs4 and ETHs6 were subjected tofluorescence in situ hybridization (FISH) (Solinas Toldo, et al., 1993)or direct in situ chromosomal PCT (DISC PCR) on porcine metaphases.Metaphase chromosomes were Q-banded and photographed beforehybridization. The probes were labeled by random priming usingbiotin-16-dUTP. Signal detection and amplification was performed usingavidin-FITC and biotinylated anti-avidin. The chromosomes werecounterstained with 4,6-diamidino-2-phenylindole, and the relativepositions of the cosmids were determined as described by Solinas Toldo,1993.

4. Subcloning

Enzymatic digests of probe positive genomic colonies were separated onagaro se gel, transferred to a nylon membrane, and probe positive bandswere subcloned into plasmids for FUT1 sequencing. The sequence of FUT1derived from this method is shown in FIG. 1.

KspI-, EcoRI- and KspI/EcoRI digests of all cosmids were separated on a0.8% agarose gel and transferred to a Hybond N nylon membrane.(Meijerink et al., 1997). This blot was hybridized with α³²P dATPlabeled porcine FUT1 PCR products (primers P6-P11 and P7-P10). Based onthe autoradiographic signals, ETHs1, −s2 and −s3 were subjected tofurther subcloning into pBluescript SK—(Stratagene), and FUT sequenceswere determined from subclones. The sequences of two FUT-like openreading frames (ORFs) (FUT1 and FUT2) obtained from cosmids ETHs2 and−s3 were compared in ECF18R positive (BB/Bb) and negative (bb) animalsby direct sequencing of PCR products.

5. Polymerase Chain Reaction and Direct Sequencing

Using the Perkin Elmer Ready Reaction Dye Terminator kit (Perkin ElmerCetus, Norwalk, Conn., USA) and 10 pmol of primer, cycle sequencing wasperformed with a thermal program consisting of an initial denaturationof 5 min at 95° C., followed by 25 cycles of 30 sec 95° C., 15 sec 50°C. and 4 min 60° C. Primers used for amplification and sequencing of theporcine alpha (1,2) fucosyltransferase genes are listed in Table 1.Additional primers were designed taking the possibility ofcross-annealing of primers due to the high similarity of FUT1, FUT2 andthe FUT2 pseudogene into account. Samples were analyzed on a 373A ABIsequencer (Applied Biosystems, Inc.) and sequence analysis was performedwith the GCG package (Devereux, 1984).

6. Production of Informative Offspring

Single nucleotide polymorphisms were analyzed in 221 Landrace swineproduced from 4 boars and 16 sows, and in 29 Large White swine producedfrom 9 matings between unrelated swine. In order to produce a largenumber of informative offspring for the examination of linkage betweenporcine genes encoding ECF18 receptors and selected polymorphic loci,only informative Landrace matings of the type B/b×b/b were produced.

7. Colonization Test

In a study of Bertschinger et al., 1993, the above mentioned Landraceswine were also tested for ECF18 susceptibility in a colonization test.For this, swine were inoculated shortly after weaning with bacteria ofE. coli strain 124/76 of serotype 0139:K12(B):H1:F18. (Rippinger, et a.,1995). Faecal shedding of the bacteria was monitored daily. The extentof colonization was calculated as the mean of the two highest faecalscores. Swine with a mean faecal score of 3.5, corresponding to 6.7 logcolony forming units (CFU)/g or more, were considered susceptible tocolonization. This limit was based on a lack of mortality below thisvalue, and on scores obtained from completely resistant litters.

8. Linkage Analysis of Nucleotide Polymorphisms

The results of the single nucleotide polymorphisms were compared withtyping data for ECF18R, which were identified in an in vitro adhesionassay described by Vögeli et al., (1996), and with typing data for theGPI-, PGD-, α-1-B-glycoprotein-(AIBG), ryanodine receptor (RYRI), EAH-and S-loci as published by Vögeli et al., (1996). Pairwise linkageanalysis and calculation of recombination fractions was performed usingthe CRI-MAP version 2.4 programme (Green et al., 1990). Multipointlinkage analysis was performed by sequential insertion of the above lociinto the map. Haplotype frequencies were calculated from the parentalanimals, in the Landrace families and from the 8 parental Large Whiteanimals which were haplotyped for from progeny information. Tetrachroiccorrelations of ECF18R and mutations in FUT1 (FUT1/M307) (polymorphisms)were calculated on all Landrace and Large White progeny.

9. Southern Blot Analysis

Southern blot analysis was performed on cosmids ETHs (1-3), ETHs2 (4-6)and ETHs3 (7-9) after digestion with enzymes KspI (1,4,7), EcoRI (2, 5,8) and KspI/EcoRI (3, 6, 9) and separation on 0.8% agarose.Hybridization with an α32 PdATP labeled 5′ FUT1 fragment (primersP6-P11) results in the same hybridizing 940 bp band in both the KspIdigest (lane 4) and the KpsI/EcoRI digest (lane 6). However,hybridization with a 3′ FUT1 fragment (primers P7-P10) (Table 1) shows a6.2 kb KspI band in lane 4 and a 1.1 kb KspI/EcoRI band in lane 6. Boththe 5′ and 3′ FUT1 fragments hybridize to the same 4.6 kb EcoRI fragmentin lane 5. This indicates the presence of a KspI site in the FUT1 genecontained in cosmid ETHs2. Cross hybridization of the 3′ FUT1 fragmentdetects 2.7 kb (lanes 2, 3, 8 and 9) and 8.2 kb (lanes 8 and 9) bands,resulting in the identification of the FUT2 pseudogene (incomplete ORF)and the FUT2 gene sequences, respectively.

10. Restriction Fragment Polymorphisms

Detection of (A) the M307 G to A and (B) the M857 G to A mutation in theporcine FUT1 gene was achieved by restriction length polymorphismanalysis, rising various restriction enzymes. Digestion of amplifiedFUT1 fragments with CfoI (A) and AciI (B) results in a restrictionfragment polymorphism. In the first lane is a 100 bp marker. Fragmentlengths are indicated in base pairs. (A) The M307^(A/A) genotype (lane2) generates 328 and 93 bp restriction fragments while the M307^(G/G)genotype (lane 4) generates 93, 241 and 87 bp fragments and heterozygousM307^(G/G) genotypes (lane 3) shows all four fragments.

(B) Digestion of the M857^(A/A) genotype (lane 2) generates 174 bpfragments, while it ggenerates 136 and 38 bp fragments in the M857^(G/G)genotypes (lane 4), and in M857^(A/G) genotypes (lane 3) all threefragments are generated.

11. Source of Swine

Data of the Swiss Landrace experimental population came from twopedigrees, which were built up at the Institute of VeterinaryBacteriology, University of Zurich. All other pigs of the Large White,Swiss Landrace, Duroc, Hampshire and Pietrain breeds came from differentbreeding herds of Switzerland. Other swine were randomly obtained fromfarms in the U.S. Midwest.

Documents Cited

-   -   Bertschinger et al. (1993) Veterinary Microbiology 35:79-89.    -   Cohney et al. (1996) Immunogenetics 44:76-79.    -   Devereux et al. (1984) Nucleic Acids Res. 1:387-395.    -   Fujii et al. (1991) Science 253:448-451.    -   Green et al. (1990) Documentation for CRI-MAP, Version 2.4, St.        Louis: Washington University School of Medicine.    -   Kelly et al. (1994), Proc. Natl. Acad. Sci., U.S.A.        91:5843-5847.    -   Meijerink, E. et al. (1997), 25^(th) Int. Conf. on Animal        Genetics, p. 44.    -   Rippinger, et al. (1995) Vet. Microbial. 45:281-295.    -   Solinas Toldo et al. (1993) Mamm. Genome 4:720-727.    -   Thurin and Blaszczyk-Thurin, (1995) J. Biol. Chem.        270(44):26577-26580.    -   Vögeli et al. (1996) Animal Genetics 27:321-328.    -   Vögeli et al. (1997) Schweiz. Arch. Tierheilk. 139:479-484.    -   U.S. Pat. No. 5,358,649, Maclennon et al.    -   U.S. Pat. No. 5,552,144, Valery et al.    -   WO 8604604 987P, Inventor: Peterson.    -   WO 9628967, Inventor: Koike, C.    -   WO 9413811, Inventors: Imberechts and Lintermans.    -   TW 266264, Inventors: Jeng and Liou.

1. A method for identifying a swine that is resistant to intestinalcolonization by toxigenic E. coli that are capable of adhering tointestinal walls of swine and causing intestinal disorders in swine,said method comprising: (a) determining whether a swine has a geneticpolymorphism, in both alleles wherein the polymorphism comprises anitrogen base adenine at position 307 in the open reading frame of thealpha (1, 2) fucosyltransferase 1 gene (FUT1) (SEQ ID NO: 12), or apolymorphism in allelic association with the FUT1 polymorphism that hasonly adenine at position 307; and (b) inferring that the swine isresistant if the swine only has adenine at position 307 or is homozygousfor a polymorphism in allelic association with FUT1 adenine in position307.
 2. A method for breeding swine that are resistant to diseasescaused by toxigenic E. coli capable of adhering to intestinal walls ofswine and causing intestinal disorders in swine, said method comprising:(a) selecting for breeding swine that are homozygous for a geneticpolymorphism in the open reading frame of the alpha (1, 2)fucosyltransferase 1 gene, wherein a nitrogen base at position 307 inthe open reading frame of the alpha (1, 2) fucosyltransferase 1 gene(SEQ ID NO: 12) of the swine is adenine, or for a polymorphism inallelic association with the FUT1 polymorphism that has adenine atposition 307; and (b) breeding the selected swine.
 3. The method ofclaim 2 wherein the E. coli is strain F18.